Rabbit anti human intrinsic factor serum was generously provided by Dr. David H. Alpers. Rabbit anti porcine megalin was described previously. Antiserum to ammnionless kinase inhibitor Imatinib was prepared by immunizing rabbits with synthetic multiple antigenic peptide con taining amino acid residues 165 178. The resulting Inhibitors,Modulators,Libraries antiserum reacted with a single 45 kDa polypeptide in mouse kidney extracts and 35 kDa and 30 kDa bands in intestinal extracts and an 30 kDa band in extracts of E8. 5 mouse embryo ex tracts and rat BN cells. Donkey anti goat and donkey anti rabbit Alexa Fluor conjugates were purchased Inhibitors,Modulators,Libraries from Invitrogen. Tissue procurement and immunofluorescence Following euthanization, animals were perfused first with phosphate buffered saline and then with 4% paraformaldehyde, PBS.
Tissues were dissected and fur ther fixed by immersion in 4% paraformaldehyde PBS for 12 h. For kidney immunohistochemical analysis, 0. 25 cm thick strips of cortex tissue were isolated. For small intestine immunohistochemical analysis, 10 cm long segments of duodenum, jejunum and ileum were isolated. Isolated tissues were Inhibitors,Modulators,Libraries embed ded in paraffin and sectioned at 6 um thickness. Tissue sections were incubated with primary antibodies diluted in PBS containing 3% BSA, washed with PBS and incubated with Alexa Fluor conjugated secondary antibodies. Nuclei were stained using Draq5. Labeled sections were analyzed using a Leica SP5 confocal microscope using the 63�� objective or a Zeiss Axio M2 microscope using the 40�� objective. Whole mount images of unlabeled intestine EGFP fluorescence were taken by a Leica MZ FLIII microscope at 5�� magnification.
Intestinal villi from Cubn del exon 1 6.EGFP mouse ileum were micro dissected under GFP light at 10�� mag nification using Leica MZ FLIII microscope. Villi that appeared predominantly EGFP positive or EGFP negative as well Inhibitors,Modulators,Libraries as a random sampling of both were separately iso lated using dissection scissors in Hanks buffered salt solu tion containing 4% FBS. Villi were then briefly span down, the supernatant removed and RNA extracted from the samples using the RNeasy Plus Mini Inhibitors,Modulators,Libraries Kit. qPCR was performed as described below. Immunoblot analysis Unfixed segments of small intestine were homogenized in 1% Triton X 100, 0. 5% Tween20, 0. 5 M NaCl, 50 mM Hepes, pH 7. 5 containing a protease inhibitor cocktail useful handbook using a Polytron aggregate. Extracts were subjected to centrifugation at 100 K g for 30 min at 4 C. Protein concentration in extracts was quantified using Pierce BCA Protein Assay Kit. Equal amounts of protein from the extracts were loaded onto NuPAGE 4 12% polyacrylamide gradient, Bis Tris gels in the presence of SDS.